High resolution x-ray structure of tyvelose epimerase from Salmonella typhi

Tyvelose epimerase catalyzes the last step in the biosynthesis of tyvelose by converting CDP-d-paratose to CDP-d-tyvelose. This unusual 3,6-dideoxyhexose occurs in the O-antigens of some types of Gram-negative bacteria. Here we describe the cloning, protein purification, and high-resolution x-ray crystallographic analysis of tyvelose epimerase from Salmonella typhi complexed with CDP. The enzyme from S. typhi is a homotetramer with each subunit containing 339 amino acid residues and a tightly bound NAD+ cofactor. The quaternary structure of the enzyme displays 222 symmetry and can be aptly described as a dimer of dimers. Each subunit folds into two distinct lobes: the N-terminal motif responsible for NAD+ binding and the C-terminal region that harbors the binding site for CDP. The analysis described here demonstrates that tyvelose epimerase belongs to the short-chain dehydrogenase/reductase superfamily of enzymes. Indeed, its active site is reminiscent to that observed for UDP-galactose 4-epimerase, an enzyme that plays a key role in galactose metabolism. Unlike UDP-galactose 4-epimerase where the conversion of configuration occurs about C-4 of the UDP-glucose or UDP-galactose substrates, in the reaction catalyzed by tyvelose epimerase, the inversion of stereochemistry occurs at C-2. On the basis of the observed binding mode for CDP, it is possible to predict the manner in which the substrate, CDP-paratose, and the product, CDP-tyvelose, might be accommodated within the active site of tyvelose epimerase.     

High resolution mapping of sediment organic matter from acoustic reflectance data

Spatial mapping of the marine environment is challenging when the properties concerned are difficult to measure except by shore-based analysis of discrete samples of material, usually from sparsely distributed sites. This is the case for many seabed sediment properties. We developed an indirect approach to mapping the organic content of coastal sediments from hydro-acoustic reflectance data. The basis was that both organic matter and acoustic reflectance are related to sediment type and grain size composition. Hence there is a collateral relationship between organic matter content and reflectance properties which can be exploited to enable high resolution mapping. We surveyed an area of seabed off the east coast of Scotland using a vessel mounted single beam echosounder with RoxAnn signal processing. Organic carbon, nitrogen and phytoplankton pigment contents were then measured in material from grab and core samples collected at intervals over a year. Relationships between the organic components and hydro–acoustic characteristics were derived by general additive models, and used to construct high resolution maps from the acoustic survey data. Our method is an advance on traditional interpolation techniques sparse spatial data, and represents a generic approach that could be applied to other properties.     

Photoisomerization mechanism of rhodopsin and 9-cis-rhodopsin revealed by x-ray crystallography

The primary photochemical process of the visual function has been investigated using the three crystallographic models, 11-cis-rhodopsin, all-trans-bathorhodopsin, and the artificial isomeric 9-cis-rhodopsin. Detailed examination of the atomic displacements and dihedral angle changes of the retinal chromophore involved in the interconversion among these isomers suggests the mechanism of isomerization efficiency.     

Electron crystallography of bacteriorhodopsin with millisecond time resolution

The goal of time-resolved crystallographic experiments is to capture dynamic "snapshots" of molecules at different stages of a reaction pathway. In recent work, we have developed approaches to determine determined light-induced conformational changes in the proton pump bacteriorhodopsin by electron crystallographic analysis of two-dimensional protein crystals. For this purpose, crystals of bacteriorhodopsin were deposited on an electron microscopic grid and were plunge-frozen in liquid ethane at a variety of times after illumination. Electron diffraction patterns were recorded either from unilluminated crystals or from crystals frozen as early as 1 ms after illumination and used to construct projection difference Fourier maps at 3.5-A resolution to define light-driven changes in protein conformation. As demonstrated here, the data are of a sufficiently high quality that structure factors obtained from a single electron diffraction pattern of a plunge-frozen bacteriorhodopsin crystal are adequate to obtain an interpretable difference Fourier map. These difference maps report on the nature and extent of light-induced conformational changes in the photocycle and have provided incisive tools for understanding the molecular mechanism of proton transport by bacteriorhodopsin.     

High resolution x-ray analysis of two mutants of a curaremimetic snake toxin.

A previous mutational analysis of erabutoxin a (Ea), a curaremimetic toxin from sea snake venom, showed that the substitutions S8G and S8T caused, respectively, 176-fold and 780-fold affinity decreases for the nicotinic acetylcholine receptor (AchR). In view of the fact that the side-chain of Ser8 is buried in the wild-type toxin, we wondered whether these affinity changes reflect a direct binding contribution of S8 to the receptor and/or conformational changes that could have occurred in Ea as a result of the introduced mutations. To approach this question, we solved X-ray structures of the two mutants S8G and S8T at high resolution (0.18 nm and 0.17 nm, with R factors of 18.0% and 17.9%, respectively). The data show that none of the mutations significantly modified the toxin structure. Even within the site where the toxin binds to the receptor the backbone conformation remained unchanged. Therefore, the low affinities of the mutants S8T and S8G cannot be explained by a large conformational change of the toxin structure. Although we cannot exclude the possibility that undetectable structural changes have occurred in the toxin mutants, our data support the view that, although buried between loop I and II, S8 is part of the functional epitope of the toxin.     

Sequencing a protein by x-ray crystallography. II. Refinement of yeast hexokinase B co-ordinates and sequence at 2.1 A resolution.

Although the amino acid sequence of yeast hexokinase B has not been determined by chemical means, crystallographic refinement of the hexokinase monomer was carried out at 2.1 Å resolution to improve both the atomic co-ordinates and the amino acid sequence, which had been obtained from a 2.5 Å electron density map. The atomic co-ordinates were adjusted by real-space refinement into a multiple isomorphous replacement map, followed by automated difference Fourier refinement, and restrained parameter structure factor least-squares refinement. The amino acid sequence was altered periodically after visual inspection of (F_o ? F_c) difference electron density maps. Evidence of the improvement in the amino acid sequence was provided by the better agreement between the X-ray and chemically derived amino acid compositions, and most importantly by the ability to locate two short peptides which had been chemically sequenced. While only 6 out of the 18 residues in these two peptides agree with the sequence of the original model, 12 residues agree with the sequence of the refined model and the others differ by only an atom or two. The refined model contains 3293 of of the 3596 non-hydrogen atoms expected from the amino acid composition and 152 bound water molecules. The crystallographic R factor at 2.1 Å is 0.25.We show that there are several advantages to refining the structure of even a protein of unknown sequence. (1) Improved phases can be obtained to the resolution limit of the diffraction pattern starting with a model derived from a 2.5 Å map. (2) The accuracy of the amino acid sequence derived by X-ray methods alone can be substantially improved. (3) Functionally important residues can be identified before chemical sequence information is available. (4) The improved X-ray sequence should greatly reduce the effort required to obtain a chemical sequence; since peptides as short as eight or nine residues can be located in the refined X-ray sequence, peptides do not need to be overlapped by chemical means.     

Crystallization of soluble proteins in vapor diffusion for x-ray crystallography

The preparation of protein single crystals represents one of the major obstacles in obtaining the detailed 3D structure of a biological macromolecule. The complete automation of the crystallization procedures requires large investments in terms of money and labor, which are available only to large dedicated infrastructures and is mostly suited for genomic-scale projects. On the other hand, many research projects from departmental laboratories are devoted to the study of few specific proteins. Here, we try to provide a series of protocols for the crystallization of soluble proteins, especially the difficult ones, tailored for small-scale research groups. An estimate of the time needed to complete each of the steps described can be found at the end of each section.     

Conformational flexibility of the acetylcholinesterase tetramer suggested by x-ray crystallography.

Acetylcholinesterase, a polymorphic enzyme, appears to form amphiphilic and nonamphiphilic tetramers from a single splice variant; this suggests discrete tetrameric arrangements where the amphipathic carboxyl-terminal sequences can be either buried or exposed. Two distinct, but related crystal structures of the soluble, trypsin-released tetramer of acetylcholinesterase from Electrophorus electricus were solved at 4.5 and 4.2 A resolution by molecular replacement. Resolution at these levels is sufficient to provide substantial information on the relative orientations of the subunits within the tetramer. The two structures, which show canonical homodimers of subunits assembled through four-helix bundles, reveal discrete geometries in the assembly of the dimers to form: (a) a loose, pseudo-square planar tetramer with antiparallel alignment of the two four-helix bundles and a large space in the center where the carboxyl-terminal sequences may be buried or (b) a compact, square nonplanar tetramer that may expose all four sequences on a single side. Comparison of these two structures points to significant conformational flexibility of the tetramer about the four-helix bundle axis and along the dimer-dimer interface. Hence, in solution, several conformational states of a flexible tetrameric arrangement of acetylcholinesterase catalytic subunits may exist to accommodate discrete carboxyl-terminal sequences of variable dimensions and amphipathicity.     

ADP-binding site of Escherichia coli succinyl-CoA synthetase revealed by x-ray crystallography

Succinyl-CoA synthetase (SCS) catalyzes the following reversible reaction via a phosphorylated histidine intermediate (His 246alpha): succinyl-CoA + P(i) + NDP <--> succinate + CoA + NTP (N denotes adenosine or guanosine). To determine the structure of the enzyme with nucleotide bound, crystals of phosphorylated Escherichia coli SCS were soaked in successive experiments adopting progressive strategies. In the first experiment, 1 mM ADP (>15 x K(d)) was added; Mg(2+) ions were omitted to preclude the formation of an insoluble precipitate with the phosphate and ammonium ions. X-ray crystallography revealed that the enzyme was dephosphorylated, but the nucleotide did not remain bound to the enzyme (R(working) = 17.2%, R(free) = 22.8% for data to 2.9 A resolution). Catalysis requires Mg(2+) ions; hence, the "true" nucleotide substrate is probably an ADP-Mg(2+) complex. In the successful experiment, the phosphate buffer was exchanged with MOPS, the concentration of sulfate ions was lowered, and the concentrations of ADP and Mg(2+) ions were increased to 10.5 and 50 mM, respectively. X-ray diffraction data revealed an ADP-Mg(2+) complex bound in the ATP-grasp fold of the N-terminal domain of each beta-subunit (R(working) = 19.1%, R(free) = 24.7% for data to 3.3 A resolution). We describe the specific interactions of the nucleotide-Mg(2+) complex with SCS, compare these results with those for other proteins containing the ATP-grasp fold, and present a hypothetical model of the histidine-containing loop in the "down" position where it can interact with the nucleotide approximately 35 A from where His 246alpha is seen in both phosphorylated and dephosphorylated SCS.     

Noncompetitive antibody neutralization of IL-10 revealed by protein engineering and x-ray crystallography

IL-10 is a dimeric cytokine that must engage its high-affinity cell surface receptor, IL-10R1, to induce multiple cellular activities. Here we report the 1.9 A crystal structure of an engineered IL-10 monomer (IL-10M1) in complex with a neutralizing Fab fragment (9D7Fab). 9D7Fab and IL-10R1 bind distinct nonoverlapping surfaces on IL-10M1. Antagonism of the IL-10M1/IL-10R1 interaction is the result of 9D7Fab-induced conformational changes in the CD loop of IL-10M1 that indirectly alter the structure of the IL-10R1 binding site. A single mutation (Ile87Ala) in the same CD loop region of the Epstein-Barr virus IL-10 (ebvIL-10) also reduces IL-10R1 binding affinity, suggesting that ebvIL-10 and 9D7Fab use similar allosteric mechanisms to modulate IL-10R1 affinity and biological activity.     

High resolution neutron fibre diffraction data on hydrogenated and deuterated cellulose

High-resolution fibre neutron diffraction data were recorded from cellulose samples on a D19 diffractometer at the Institut Laue-Langevin (Grenoble). Highly crystalline cellulose I samples from Cladophora (cellulose I alpha + I beta) or Halocynthia (cellulose I beta) origin were prepared in the form of oriented films. Samples were studied in a hydrogenated form and in a hydrogen-deuterium exchanged deuterated form corresponding to all OH moieties being replaced by ODs. These samples, which diffracted to a resolution of around 0.9 A, gave diffraction diagrams consisting of several hundred independent diffraction spots. Crystalline cellulose II fibres resulting from the mercerization of flax were also studied in a hydrogenated form using NaOH/H2O as mercerizing medium and in a deuterated form using NaOD/D2O. Both of these samples diffracted to around 1.2 A, giving fibre diffraction diagrams slightly less resolved than those of cellulose I, but still consisting of more than one hundred independent diffraction spots. For cellulose I as well as for cellulose II, significant differences between the hydrogenated and deuterated patterns were observed and recorded. These new data should lead to improved structures for cellulose and direct identification of the position of hydrogen atoms involved in hydrogen bonding.     

High pressure nmr study of dihydrofolate reductase from a deep-sea bacterium Moritella profunda.

We have investigated the effect of pressure and temperature on the structural and thermodynamic stability of a protein dihydrofolate reductase from a deep-sea bacterium Moritella profunda in its folate-bound form in the pressure range between 3 and 375 MPa and the temperature range between -5 and 30 degrees C. The on-line cell variable pressure 1H NMR spectroscopy has been used to analyze the chemical shift and signal intensity in one-dimensional 1H NMR spectra. Thermodynamic analysis based on signal intensities from protons in the core part indicates that the thermodynamic stability of Moritella profunda DHFR is relatively low over the temperature range between -5 and 30 degrees C (deltaG0=15.8 +/- 4.1 kJ/mol at 15 degrees C), but is well adapted to the living environment of the bacterium (2 degrees C and 28 MPa), with the maximum stability around 5 degrees C (at 0.1 MPa) and a relatively small volume change upon unfolding (deltaV= 66 +/- 19 ml/mol). Despite the relatively low overall stability, the conformation in the core part of the folded protein remains intact up to approximately 200 MPa, showing marked stability of the core of this protein.     

Isolation and characterization of proteases from Euphorbia lactea and Euphorbia lactea cristata

Latex from E. lactea yielded three homogeneous proteases, euphorbains, 1a₁, 1a₂ and 1a₃ with M_r of 66 k, 44 k and 33 k respectively. Euphorbains 1a₁ and 1a₃ had unique pIs of, in order, 7.0 and 4.5, while 1a₂ comprised three charged forms with pIs ranging from 5.0 to 6.4. From the latex of E. lactea cristata a single proteolytic euphorbain 1c was isolated which had an M_r of 70 000 and five pIs between 5.0 and 8.0. Euphorbains 1a₁ and 1c have similar substrate specificities which are different from those of 1a₂ and 1a₃. Euphorbains 1a₁, 1a₂ and 1c are serine-centred enzymes with vital histidine residues, and the latter protease is activated by Ca²⁺, Mg²⁺ and Mn²⁺.     

Simulation-based methods for interpreting x-ray data from lipid bilayers

The fully hydrated liquid crystalline phase of the dimyristoylphosphatidycholine lipid bilayer at 30 degrees C was simulated using molecular dynamics with the CHARMM potential for five surface areas per lipid (A) in the range 55-65 A(2) that brackets the previously determined experimental area 60.6 A(2). The results of these simulations are used to develop a new hybrid zero-baseline structural model, denoted H2, for the electron density profile, rho(z), for the purpose of interpreting x-ray diffraction data. H2 and also the older hybrid baseline model were tested by fitting to partial information from the simulation and various constraints, both of which correspond to those available experimentally. The A, rho(z), and F(q) obtained from the models agree with those calculated directly from simulation at each of the five areas, thereby validating this use of the models. The new H2 was then applied to experimental dimyristoylphosphatidycholine data; it yields A = 60.6 +/- 0.5 A(2), in agreement with the earlier estimate obtained using the hybrid baseline model. The electron density profiles also compare well, despite considerable differences in the functional forms of the two models. Overall, the simulated rho(z) at A = 60.7 A(2) agrees well with experiment, demonstrating the accuracy of the CHARMM lipid force field; small discrepancies indicate targets for improvements. Lastly, a simulation-based model-free approach for obtaining A is proposed. It is based on interpolating the area that minimizes the difference between the experimental F(q) and simulated F(q) evaluated for a range of surface areas. This approach is independent of structural models and could be used to determine structural properties of bilayers with different lipids, cholesterol, and peptides.     

Automated crystal mounting and data collection for protein crystallography

To increase the efficiency of diffraction data collection for protein crystallographic studies, an automated system designed to store frozen protein crystals, mount them sequentially, align them to the X-ray beam, collect complete data sets, and return the crystals to storage has been developed. Advances in X-ray data collection technology including more brilliant X-ray sources, improved focusing optics, and faster-readout detectors have reduced diffraction data acquisition times from days to hours at a typical protein crystallography laboratory [1,2]. In addition, the number of high-brilliance synchrotron X-ray beam lines dedicated to macromolecular crystallography has increased significantly, and data collection times at these facilities can be routinely less than an hour per crystal. Because the number of protein crystals that may be collected in a 24 hr period has substantially increased, unattended X-ray data acquisition, including automated crystal mounting and alignment, is a desirable goal for protein crystallography. The ability to complete X-ray data collection more efficiently should impact a number of fields, including the emerging structural genomics field [3], structure-directed drug design, and the newly developed screening by X-ray crystallography [4], as well as small molecule applications.